Overview of general physiologic features and functions of vitamin D1 4

Hector F DeLuca
impart vitamin D and rickets as a major medical problem would
disappear. Second, the irradiation of fat-soluble substances extracted from tissues could be used to generate large amounts of
vitamin D for later characterization. The structure of vitamin D2
was deduced in 1931 by Askew et al (9), and the structure of
vitamin D3 was determined through synthetic means by Windaus
et al (10). Vitamin D was discovered with many other vitamins
and is classed as a vitamin even now. However, findings from the
second half of the 20th century showed that vitamin D is truly a
prohormone and not a vitamin. Vitamin D is virtually absent from
the food supply. It is not found in plant materials (eg, vegetables,
fruits, or grains) and is present in low abundance in meats and
other animal food sources, except in rare cases such as fish liver
oils and plants such as waxy-leaf nightshade (Solanum glaucophyllum).

KEY WORDS
Vitamin D metabolism, bone, calcium homeostasis, tetany, vitamin D endocrine system, autoimmune diseases
PRODUCTION AND METABOLISM OF VITAMIN D
INTRODUCTION

The discovery of vitamin D and the elimination of rickets as a

major medical problem must rank as one of medicines great
achievements (1). From the early studies of McCollum and Davis
(2) in 1913, when the first vitamin was discovered, until 1940, the
work leading to the identification of vitamin D and its role in bone
formation and prevention of hypocalcemic tetany included many
outstanding contributions. Most noteworthy was the work by Sir
Edward Mellanby, who demonstrated that he could produce rickets in dogs by feeding them the diet characteristic of Scotland, ie,
oatmeal; unknown to Sir Edward Mellanby was the fact that he
deprived those dogs of sunlight. Because of the work of McCollum and Davis in discovering fat-soluble vitamin A, Mellanby
attributed the ability of cod liver oil to cure the rachitic condition
in dogs as being another property of vitamin A (3). McCollum
very cleverly destroyed the vitamin A activity of cod liver oil by
bubbling oxygen through the solution and heating it, but the
ability to cure rickets remained in the preparation. McCollum
correctly concluded that this represented a new vitamin, called
vitamin D (4). Huldshinsky (5) and Chick et al (6) independently
demonstrated that rachitic children could be cured with exposure
to sunlight or artificially produced ultraviolet light. The puzzle
was ultimately solved when Steenbock and Black (7) discovered
that irradiation not only of the skin of animals but also of the food
they consumed imparted antirachitic activity to either the animals or their food. Furthermore, Goldblatt and Soames (8)
showed that livers taken from irradiated rats could heal rickets in
rats. Therefore, 2 important discoveries occurred. First, Steenbock and Black (7) conceived that foods could be irradiated to

Vitamin D is normally produced in skin through a robust

photolytic process acting on a derivative of cholesterol (ie,
7-dehydrocholesterol) to produce previtamin D, which is then
slowly isomerized to vitamin D3 (11). Vitamin D3 is the natural
form of vitamin D produced in skin, and vitamin D2 is derived
from irradiation of ergosterol, which occurs to some degree in
plankton under natural conditions and is used to produce vitamin
D2 from the mold ergot (which contains as much as 2% ergosterol). We must move away from the concept that vitamin D is a
vitamin.
Another important fact is that vitamin D is required throughout
life. It not only is needed for the formation of bone but also likely
plays an important role in several other physiologic systems. Its
use may well prevent several degenerative diseases, and it may
also play a role as an anticancer agent.
The structure of vitamin D3 and its numbering system are
indicated in Figure 1. We now know that vitamin D3 itself is
biologically inert, as clearly indicated by genetic defects that
result in the disease rickets despite normal intakes of vitamin D
(12). By 1967, the concept that vitamin D is converted to an
active form had appeared (13, 14). By 1969, the circulating form
1
From the Department of Biochemistry, University of WisconsinMadison.
2
Presented at the conference Vitamin D and Health in the 21st Century:
Bone and Beyond, held in Bethesda, MD, October 9 10, 2003.
3
Supported in part by the Wisconsin Alumni Research Foundation.
4
Address correspondence to HF DeLuca, Department of Biochemistry,
University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 537061544. E-mail: deluca@biochem.wisc.edu.

Am J Clin Nutr 2004;80(suppl):1689S96S. Printed in USA. 2004 American Society for Clinical Nutrition

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ABSTRACT
Vitamin D3 is a prohormone produced in skin through ultraviolet
irradiation of 7-dehydrocholesterol. It is biologically inert and must
be metabolized to 25-hydroxyvitamin D3 in the liver and then to
1,25-dihydroxyvitamin D3 in the kidney before function. The hormonal form of vitamin D3, ie, 1,25-dihydroxyvitamin D3, acts
through a nuclear receptor to carry out its many functions, including
calcium absorption, phosphate absorption in the intestine, calcium
mobilization in bone, and calcium reabsorption in the kidney. It also
has several noncalcemic functions in the body. This overview provides a brief description of the physiologic, endocrinologic, and
molecular biologic characteristics of vitamin D. It also provides
information on new selective analogs of 1,25-dihydroyvitamin D3
for therapy.
Am J Clin Nutr 2004;80(suppl):1689S96S.

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FIGURE 1. Structure of vitamin D3, or cholecalciferol, and its numbering

system.

FIGURE 2. Metabolic activation of vitamin D3 to its hormonal form,

1,25(OH)2D3.

rickets type I were identified in several studies (20). Very important was the generation of 1-hydroxylase knockout mice,
which exhibit a phenotype virtually identical to the human vitamin D-dependent rickets type I phenotype. Therefore, the enzymes that activate vitamin D have been identified.
Of major metabolic importance is the mode of disposal of
vitamin D and its hormonal forms. The cytochrome P-450 enzyme now called CYP24 was isolated in pure form by Ohyama
and Okuda (23) and the complementary DNA and gene were
cloned, which yielded a 24-hydroxylase-null mutant (reviewed
in 20). No significant phenotype resulted except for a large accumulation of 1,25(OH)2D3 in the circulation, which produced
secondary effects on cartilaginous growth (20, 24). CYP24 is an
extremely active enzyme, but the gene remains silent in vitamin
D deficiency; it is induced by the hormonal form of vitamin D
itself. Therefore, pulses of the vitamin D hormone program its
own death through induction of the 24-hydroxylase. The 24hydroxylase is able to metabolize vitamin D to its excretion
product calcitroic acid (20). 25(OH)D3 can also be degraded
through this pathway. 24-Hydroxylase and its regulation are
important factors in the determination of the circulating concentrations of the hormonal form of vitamin D.
PHYSIOLOGIC FUNCTIONS OF VITAMIN D

A diagrammatic explanation of the role of the vitamin D hormone in mineralizing the skeleton and preventing hypocalcemic
tetany is presented in Figure 3 (20). Plasma calcium concentrations are maintained at a very constant level, and this level is
supersaturating with respect to bone mineral. If the plasma becomes less than saturated with respect to calcium and phosphate,
then mineralization fails, which results in rickets among children
and osteomalacia among adults (24). The vitamin D hormone
functions to increase serum calcium concentrations through 3
separate activities. First, it is the only hormone known to induce
the proteins involved in active intestinal calcium absorption.
Furthermore, it stimulates active intestinal absorption of phosphate. Second, blood calcium concentrations remain in the normal range even when an animal is placed on a no-calcium diet.
Therefore, an animal must possess the ability to mobilize calcium
in the absence of calcium coming from the environment, ie,

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of vitamin D had been isolated, chemically identified, and synthesized (15, 16). This compound, 25-hydroxyvitamin D3
[25(OH)D3], is now currently monitored in serum to indicate the
vitamin D status of patients, as discussed below. However,
25(OH)D3 itself is metabolically inactive and must be modified
before function. The final active hormone derived from vitamin
D was isolated and identified in 1971, and its structure was
deduced as 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] (17) and
confirmed by synthesis (18). The pathway that vitamin D must
follow is illustrated in Figure 2 and forms the basis of the vitamin
D endocrine system. For 2 decades, there was consistent revisitation of the concept that more than one hormone was derived
from vitamin D, and 33 metabolites of vitamin D were identified (19). However, it soon became clear that all metabolites were
either less active or rapidly cleared and were thus intermediates
in the degradation of this important molecule. The most important of these metabolites are 24,25-dihydroxyvitamin D3 and
1,24(R),25-trihydroxyvitamin D3 produced by the enzyme
CYP24, which is induced by the vitamin D hormone itself (20).
Much is known about the enzymes that produce 1,25(OH)2D3
and their regulation, but a great deal remains to be learned (20).
Two enzymes are thought to function in the 25-hydroxylation
step. They are not exclusively hepatic but are largely functionally
active in the liver. The mitochondrial enzyme, which is not specific for vitamin D, has been cloned and a knockout mouse strain
has been prepared, without any apparent effect on vitamin D
metabolism, which suggests that there is an alternate 25hydroxylase (21). A microsomal hydroxylase was recently
cloned and could represent the missing enzyme (22). The
25(OH)D3 1-hydroxylase was cloned by 3 different laboratories (reviewed in ref 20), and the sites of vitamin D-dependent

FIGURE 3. Diagrammatic representation of the role of the vitamin D

hormone and the parathyroid hormone (PTH) in increasing plasma calcium
concentrations to prevent hypocalcemic tetany (neuromuscular) and to provide for mineralization of the skeleton.

FUNCTIONS OF VITAMIN D

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FIGURE 5. Partial list of VDREs found in the promoter regions of target

genes. Most prominent is the VDRE found in the 24-hydroxylase (CYP24)
promoter.

through enterocytes. Two mechanisms play a role in increasing

blood calcium concentrations, especially in the absence of intestinal calcium absorption. Vitamin D hormone stimulates osteoblasts to produce receptor activator nuclear factor-B ligand
(RANKL) (25). RANKL then stimulates osteoclastogenesis and
activates resting osteoclasts for bone resorption (25). Therefore,
the vitamin D hormone plays an important role in allowing individuals to mobilize calcium from bone when it is absent from
the diet. It is very important to note, however, that in vivo both
vitamin D and parathyroid hormone are required for this mobilization event (26, 27). Therefore, 2 keys are required, similar to
a safety deposit box. Third, the distal renal tubule is responsible
for reabsorption of the last 1% of the filtered load of calcium, and
the 2 hormones interact to stimulate the reabsorption of this last
1% of the filtered load (28). Because 7 g of calcium are filtered
every day among humans, this represents a major contribution to
the calcium pool. Again, both parathyroid hormone and the vitamin D hormone are required. Calcium physiologic processes
are such that a single low concentration of the vitamin D hormone
stimulates enterocytes to absorb calcium and phosphate. If the
plasma calcium concentration fails to respond, then the parathyroid glands continue to secrete parathyroid hormone, which increases production of the vitamin D hormone to mobilize bone
calcium (acting with parathyroid hormone). Under normal circumstances, environmental calcium is used first; if environmental calcium is absent, then internal stores are used.
VITAMIN D ENDOCRINE SYSTEM

A diagrammatic representation of the endocrine regulation of

calcium concentrations in the plasma and the vitamin D endocrine system is presented in Figure 4. Calcium-sensing proteins
that sense plasma calcium concentrations are found in the parathyroid gland (29, 30). When calcium concentrations decrease
below normal, even slightly, then these transmembrane proteins,
coupled to a G protein system, stimulate the secretion of parathyroid hormone. Parathyroid hormone then proceeds to the osteoblasts and to the proximal convoluted tubule cells within
seconds. Most importantly, in the convoluted tubule cells that

serve as the endocrine gland for the vitamin D hormone, 1hydroxylase concentrations are markedly elevated (30, 31). This
signals the vitamin D hormone, which by itself stimulates intestinal absorption of calcium or together with parathyroid hormone, at higher concentrations, stimulates mobilization of bone
calcium and renal reabsorption of calcium. The increase in serum
calcium concentrations exceeds the set point of the calciumsensing system, shutting down the parathyroid gland-induced
cascade of events. If the plasma calcium concentrations overshoot, then the C-cells of the thyroid gland secrete the 32-amino
acid peptide calcitonin, which blocks bone calcium mobilization
(32). Calcitonin also stimulates the renal 1-hydroxylase to provide the vitamin D hormone for noncalcemic needs under normocalcemic conditions (33). The molecular mechanisms have
not been entirely determined, except for the vitamin D hormone
induction of 24-hydroxylase (CYP24).
An important aspect of the vitamin D endocrine system is that
dietary calcium is favored to support serum calcium concentrations under normal conditions but, when this fails, the system
mediates calcium mobilization from bone and reabsorption in the
kidney to satisfy the needs of the organism. This results in loss of
calcium from the skeleton and can ultimately lead to osteoporosis. Another important aspect is that, except for stimulating mineralization of the skeleton, the vitamin D hormone has not been
found to be anabolic on bone by itself.
MOLECULAR MECHANISMS OF VITAMIN D ACTIONS

The vitamin D hormone functions through a single vitamin D

receptor (VDR), which has been cloned for several species including humans, rats, and chickens. It is a member of the class II
steroid hormones, being closely related to the retinoic acid receptor and the thyroid hormone receptor (reviewed in ref 20). It,
like other receptors, has a DNA-binding domain called the
C-domain, a ligand-binding domain called the E-domain, and an
F-domain, which is one of the activating domains. Despite many
statements to the contrary in the literature, a single receptor
appears to mediate all of the functions of vitamin D, which
complicates the preparation of analogs for one specific function
rather than another. The human receptor is a 427-amino acid
peptide, whereas the rat receptor contains 423 amino acids and
the chicken receptor contains 451 amino acids. This receptor acts
through vitamin D-responsive elements (VDREs), which are
usually found within 1 kilobase of the start site of the target gene.
The VDREs, which are shown in Figure 5, are repeat sequences

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FIGURE 4. Diagrammatic representation of the vitamin D endocrine

system. Red arrows indicate suppression; black arrows indicate stimulation.
Serum calcium concentrations are represented by a thermometer. Low serum
calcium concentrations are detected by a calcium sensor in the parathyroid
gland, which initiates synthesis and secretion of parathyroid hormone. The
calcium sensor for high concentrations is in the C cells of the thyroid gland
and initiates secretion of calcitonin (CT).

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of 6 nucleotides separated by 3 nonspecified bases. It is now clear

that the 5' arm of this sequence binds the retinoic acid X receptor
and the 3' arm binds the VDR. Of all of the genes identified to
date, the most powerfully regulated is the CYP24 or 24hydroxylase enzyme, which is responsible for the degradation of
vitamin D (20). The programming of its own destruction is thus
an important aspect of this endocrine system, which uses one of
the most potent ligands known.
A diagram that describes how the VDR with its ligand affects
the transcription of target genes is presented in Figure 6. Although there is little evidence for a co-repressor, we think that
co-repressors will eventually be found for the VDR. When the
VDR interacts with the ligand, the repressor is no longer able to
bind to the receptor, and the receptor changes conformation.
Together with the required RXR, the VDR forms a heterodimer
at the VDREs (20). At the same time, it binds several other
proteins required in the transcription complex and, most importantly, acquires an activator (20). To date, at least 3 coactivators
have been identified, ie, SARC1, -2, and -3 (34) and DRIP205
(35). There may be additional coactivators, and there may be
selectivity among the coactivators with respect to which gene is
being expressed. Much attention is being focused on this aspect
for selective regulation of target genes. Once the complex is
formed, the DNA bends (36), phosphorylation on serine-205
occurs (37), and transcription is either initiated or suppressed,
depending on the gene. To date, it is unclear whether the phosphorylation plays a functional role in transcription.
FUNCTIONS OF VITAMIN D UNRELATED TO
CALCIUM

One of the most important findings after discovery of the

receptor was that the receptor appeared not only in the target cells
of enterocytes, osteoblasts, and distal renal tubule cells but also

in parathyroid gland cells, skin keratinocytes, promyelocytes,

lymphocytes, colon cells, pituitary gland cells, and ovarian cells
(20). The expression of VDRs in these cells and not in skeletal
muscle, heart muscle, and liver suggests that they must serve a
function there (20). Although VDRs have been reported in liver,
heart, and skeletal muscle (38 42), we and other groups failed to
confirm those reports, with the use of specific monoclonal antibodies and other methods (43, 44). This led to the investigation
and discovery of functions of vitamin D not previously appreciated, which takes the vitamin D system beyond bone.
An important discovery was made by Suda et al (25), who
demonstrated that the vitamin D hormone plays an important role
in the terminal differentiation of promyelocytes to monocytes,
which are precursors of the giant osteoclasts. Those authors also
found that, when the cells differentiated into a functional cell
line, growth ceased. This function did not involve calcium and
phosphorus and was later shown to be fundamental to vitamin
D-induced production of osteoclasts through the RANKL system
(for review, see reference 25).
Of great importance is the finding of the VDR in the parathyroid glands. We now know, through the treatment of renal osteodystrophy with the vitamin D hormone and its analogs, that an
essential site for this therapy is the VDR in the parathyroid glands
(20). An important function of the vitamin D hormone is to keep
the production of the preproparathyroid gene under control and
reasonably suppressed (20, 45). Furthermore, the vitamin D hormone, through its receptor, in some way functions to prevent
proliferation of parathyroid gland cells. Therefore, an important
function of the vitamin D hormone among normal subjects is to
maintain normal parathyroid status. Among patients with renal
failure, the site of production of the vitamin D hormone is destroyed and the parathyroid gland becomes vitamin D deficient;
in the presence of adequate amounts of calcium in the circulation,

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FIGURE 6. Diagrammatic representation of the known molecular events in the regulation of gene expression by the vitamin D hormone, 1,25(OH)2D3,
acting through its receptor, VDR. The result of regulation may be either suppression or activation. RXR, retinoid X receptor; DRE, VDRE (see Figure 5); TFIIB,
transcription factor IIB; TFIID, transcription factor IID; RNAP, RNA polymerase.

FUNCTIONS OF VITAMIN D

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FIGURE 8. Structure and name of the carbon-2modified analog of

1,25(OH)2D3 that is selective in its activity on the osteoblasts of bone (2MD).

dendritic cells that present antigens to the T cells (54). Although the mechanisms of this regulation of autoimmune diseases are not understood, the results are sufficient to warrant
investigation of vitamin D analogs for the treatment of such
diseases. As an extension of this function of vitamin D, the
utility of 1,25(OH)2D3 in helping prevent transplant rejection
has been demonstrated with both vascular and nonvascular
transplants (55).
ANALOGS OF 1,25(OH)2D3

the parathyroid gland hyperproliferates and hypersecretes parathyroid hormone, resulting in secondary hyperparathyroidism
(46). An important available treatment is the use of the vitamin D
hormone and its analogs to manage the parathyroid glands of
patients undergoing dialysis.
Another important area of investigation has been the immune system. Clearly, vitamin D deficiency affects the immune system, especially T cell-mediated immunity, whereas
vitamin D in excess actually suppresses certain aspects of the
immune system (47, 48). This has led to investigation of the
use of vitamin D compounds to suppress certain autoimmune
disorders. The first autoimmune disorder to come under scrutiny is multiple sclerosis, and experimental autoimmune encephalomyelitis has been used as an animal model. This disease can be suppressed or eliminated at any stage of
development with adequate amounts of the vitamin D hormone administered orally each day (49). However, hypercalcemia occurs with this therapy. We now know that the increase
in serum calcium concentrations plays some role in this therapeutic response. A clearer example of an autoimmune disease that is regulated by the vitamin D hormone is type 1
diabetes mellitus (50). Among nonobese diabetic rats, vitamin
D deficiency caused a marked increase in incidence and a
marked decrease in the lag time required for the initiation of
diabetes (Figure 7). Very important is the fact that large doses
of the vitamin D hormone could suppress type 1 diabetes
mellitus completely (50), preventing the destruction of islet
cells. Similar results were obtained with models of systemic
lupus (51), inflammatory bowel disease (52), and rheumatoid
arthritis (53). It is likely that the suppression of these autoimmune diseases involves the vitamin D hormone interacting
with T helper lymphocytes, which in turn suppress the inflammatory responses of T helper type 1 lymphocytes. Alternative
ideas have also been put forth, such as suppression of the

We now come to the area of the design of 1,25(OH)2D3 analogs with selective activities for use against specific diseases.
The major problem is that the primary role of 1,25(OH)2D3 is to
adjust serum calcium and phosphorus concentrations. This is its
dominant role, and the design of any analog to treat a disease
other than osteoporosis or osteomalacia must include the elimination or marked suppression of the plasma calcium-increasing
activity. Most analog development in this field has been performed with that in mind. Years of experience with modifications
of 1,25(OH)2D3 and assessments of the consequent physiologic
effects have yielded some information that is very useful for
designing analogs for particular uses. A recent development has
been the understanding that the carbon-2 position of vitamin D
not only is tolerated but actually produces a much more stable
transcription complex, compared with vitamin D analogs without carbon-2 modifications (56, 57). Our group has developed
analogs that are selective for actions on osteoblasts, particularly
the anabolic or bone-forming actions of that cell type. The most
promising of the compounds studied is 2-methylene-19-nor20S-1,25-dihydroxyvitamin D3 (2MD) (Figure 8). This compound is very selective for its action on bone, being 80 100
times more effective than the native hormone in stimulating bone
calcium mobilization while being equally effective in the intestine. Demonstrating that osteoblastic activity is favored by this
analog, incubation of 2MD with human osteoblasts caused formation of bone nodules within 2 wk (Figure 9) (58). However,
incubation of the same cells with even high concentrations of
1,25(OH)2D3 produced little or no change. These results suggest
strong bone-forming activity of 2MD. To test whether this analog
could cause bone synthesis in vivo, 2MD was given to aged
female rats that had been ovariectomized to ensure a loss of bone
mass (osteoporosis). 2MD caused a marked increase in the synthesis of new bone, yielding a high bone mass value; samples
tested for breaking strength proved to be extremely strong. In the

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FIGURE 7. Time course of the development of diabetes among NOD/

LTJ female mice. Weanling mice were fed either a vitamin D-deficient diet
containing normal amounts of calcium and phosphorus or a vitamin
D-sufficient diet containing, in addition to the components of the vitamin
D-deficient diet, vitamin D3. In addition, the diet was supplemented with 50
ng of 1,25(OH)2D3 in a daily ration for the mice. The incidence of diabetes
is shown on the left, whereas the daily onset is shown on the x-axis. There is
no doubt that 1,25(OH)2D3 can prevent the onset of diabetes among NOD/Ltj
female mice.

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FIGURE 9. Incubation of human osteoblasts with 2MD, resulting in the

appearance of marked mineralized bone nodules. Osteoblasts and osteoblast
precursors harvested from pediatric bone samples discarded during surgery
were incubated at 105 cells per plate with 108 mol/L 1,25(OH)2D3, 1010 or
1012 mol/L 2MD, or no vitamin D for a period of 7 d. Fresh medium was
presented to the cells every 3 d. After 1 wk of incubation, the medium was
replaced with ascorbic acid/-glycerol phosphate-containing medium (without vitamin D compounds). After a 2-wk period, the cells were stained with
silver nitrate to reveal bone nodules (arrows).

concentrations. However, much lower doses of the same compounds are able to suppress plasma parathyroid hormone concentrations, which shows that they are systemically active.
Therefore, there is great promise that new analogs with very
selective activities can be prepared. One group of analogs is very
selective in stimulating bone synthesis. Another pair of analogs
are devoid of calcium activity while retaining systemic activity
for the parathyroid glands. The latter group might be used to treat
autoimmune diseases and cancer and to suppress secondary hyperparathyroidism among patients undergoing dialysis.
ASSESMENT OF VITAMIN D STATUS

FIGURE 10. Structures of noncalcemic, carbon-2modified analogs of

1,25(OH)2D3.

One of the most important facets of vitamin D is its many

physiologic activities. The importance of vitamin D being continually available is obvious. Vitamin D deficiencies compromise not only bone mineralization but also many other biological
activities. It is well known that vitamin D deficiency rickets is
appearing even in highly developed countries. In Europe, vitamin D fortification of foods is largely absent, and little vitamin D
is made in the skin of individuals in the northern and southern
regions of the planet during the winter months. To protect against
bone diseases and other kinds of degenerative diseases and autoimmune diseases, adequate concentrations of vitamin D are
extremely important. In the view of many scientists in the vitamin
D field, the recommended dietary allowance is too low. Supplementation with vitamin D3 at 2000 IU/d should be considered and
should be perfectly safe. To determine safety, an assessment of
vitamin D status is required. Generally, 25(OH)D3 concentrations are considered the best measure of vitamin D status. Unfortunately, commercially available assays for 25(OH)D3
yielded widely differing results (Figure 11) (60). To determine
which of these values truly represents the 25(OH)D3 concentration, we measured 25(OH)D3 concentrations in the same serum
samples with a chemical method and we found that only 2 values
agreed with the value determined chemically. Therefore, there is

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same model, 1,25(OH)2D3 administered at much higher doses

was unable to induce the same levels of bone synthesis and bone
mass. 2MD is now in phase 2 of development for osteoporosis
and appears extremely promising as an anabolic agent for bone
growth.
Two other analogs modified at carbon-2 are shown in Figure
10 (59). These compounds bind very well to the receptor and are
active in transcription but, even when given orally to animals at
doses as high as 70 g/kg, are unable to increase serum calcium

FIGURE 11. 25(OH)D3 concentrations in 20 serum samples, as measured

with assay kits from 5 different commercial laboratories and with HPLC in
our own laboratory (Lab 6). The results indicate very clearly that the laboratory values differ according to the laboratory performing the tests, some of
which are antibody-based tests. Only 2 methods (Lab 1 and Lab 3) yielded
values approaching the values obtained with the accurate chemical or HPLC
method. These results demonstrate a need for assay certification before
public health parameters can be set on the basis of 25(OH)D3 concentrations
in plasma.

FUNCTIONS OF VITAMIN D

a great need to develop a standard and highly reliable 25(OH)D3

assay before supplementation levels can be set on the basis of
serum 25(OH)D3 concentrations. It is well known that, when
large amounts of vitamin D are given to a patient, much of the
vitamin D is stored in adipose tissues. Once these sites have been
saturated, the vitamin D remains in serum and is converted to
25(OH)D3, which is toxic as an analog of 1,25(OH)2D3 (61).
When the dietary levels of vitamin D3 needed to achieve normal
concentrations of 25(OH)D3 in the plasma are being determined,
vitamin D3 itself should be measured, to confirm that vitamin D3
is not being accumulated to an extent that would result in vitamin
D intoxication. These measurements with well-established, precise methods, together with a careful, clinical, dose-escalation
study, should allow setting of a supplementation level that is safe
and can help prevent degenerative diseases, as well as preventing
or reducing the risk of autoimmune diseases.
CONCLUSIONS

HFD is president and chief executive officer of Deltanoid Pharmaceuticals

(Madison, WI) and serves as a consultant for Pfizer. Deltanoid is developing
some of the compounds described.

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14. Morri H, Lund J, Neville PF, DeLuca HF. Biological activity of a vitamin
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15. Blunt JW, DeLuca HF, Schnoes HK. 25-Hydroxycholecalciferol: a biologically active metabolite of vitamin D3. Biochemistry 1968;7:331722.
16. Blunt JW, DeLuca HF. The synthesis of 25-hydroxycholecalciferol:
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17. Holick MF, Schnoes HK, DeLuca HF, Suda T, Cousins RJ. Isolation and
identification of 1,25-dihydroxycholecalciferol: a metabolite of vitamin
D active in intestine. Biochemistry 1971;10:2799 804.
18. Semmler EJ, Holick MF, Schnoes HK, DeLuca HF. The synthesis of
1,25-dihydroxycholecalciferol: a metabolically active form of vitamin
D3. Tetrahedron Lett 1972;40:414750.
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21. Rosen H, Reshef A, Maeda N, et al. Markedly reduced bile acid synthesis but
maintained levels of cholesterol and vitamin D metabolites in mice with
disrupted sterol 25-hydroxylase gene. J Biol Chem 1998;273:1480512.
22. Cheng JB, Motola DL, Mangelsdorf DJ, Russell DW. De-orphanization
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of 25-hydroxyvitamin D3. J Biol Chem 1991;266:8690 5.
24. Underwood JL, DeLuca HF. Vitamin D is not directly necessary for bone
growth and mineralization. Am J Physiol 1984;246:E493 8.
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27. Garabedian M, Tanaka Y, Holick MF, DeLuca HF. Response of intestinal calcium transport and bone calcium mobilization to 1,25dihydroxyvitamin D3 in thyroparathyroidectomized rats. Endocrinology
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28. Yamamoto M, Kawanobe Y, Takahashi H, Shimazawa E, Kimura S,
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29. Brown EM, Gamba G, Riccardi R, et al. Cloning and characterization of
an extracellular Ca2-sensing receptor from bovine parathyroid. Nature
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30. Tanaka Y, DeLuca HF. Rat renal 25-hydroxyvitamin D3-1- and 24-hydroxylases: their in vivo regulation. Am J Physiol 1984;246:E168 73.
31. Brenza HL, DeLuca HF. Regulation of 25-hydroxyvitamin D3 1hydroxylase gene expression by parathyroid hormone and 1,25dihydroxyvitamin D3. Arch Biochem Biophys 2000;381:14352.
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with the vitamin D3 receptor in a ligand-dependent manner and enhances

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Vitamin D has yielded a class of compounds that can be used

for the treatment of a variety of diseases. Vitamin D3 itself is
converted, in a 2-step process, to 1,25(OH)2D3. This hormone
reacts with a single nuclear type 2 receptor to facilitate the activation or suppression of target genes. The proteins produced in
response to the hormone then carry out classic and nonclassic
functions of vitamin D. In addition to causing mineralization of
the skeleton and increasing serum calcium and phosphorus concentrations, vitamin D is known to regulate parathyroid growth
and parathyroid hormone production; it plays a role in the islet
cells of the pancreas, has a significant effect on the immune
system, and can help in suppression of certain autoimmune diseases and certain cancers. To obtain maximal benefits of dietary
vitamin D and to reduce the risks of these diseases, intakes of
vitamin D higher than currently recommended are in order. Furthermore, a standardized 25(OH)D3 assay that provides true values must be developed; findings could provide a basis for understanding what levels of supplementation must be used to yield
adequate amounts of 25(OH)D3.

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VDR transactivation in a cell-free system. Genes Dev 1998;12:1787

800.
Kimmel-Jehan C, Darwish HM, Strugnell SA, Jehan F, Wiefling B,
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Brown TA, DeLuca HF. Phosphorylation of the 1,25-dihydroxyvitamin
D3 receptor: a primary event in 1,25-dihydroxyvitamin D3 action. J Biol
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Fraga C, Blanco M, Vigo E, Segura C, Garcia-Caballero T, PerezFernandez R. Ontogensis of the vitamin D receptor in rat heart. Histochem Cell Biol 2002;117:54750.
Bischoff HA, Borchers M, Gudat F, et al. In situ detection of 1,25dihydroxyvitamin D3 receptor in human skeletal muscle tissue. Histochem J 2001;33:19 24.
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spinal cord. J Chem Neuroanat 1999;16:135 45.
Gascon-Barre M, Demers C, Mirshahi A, Neron S, Zalzal S, Nanci A.
The normal liver harbors the vitamin D nuclear receptor in nonparenchymal and biliary epithelial cells. Hepatology 2003;37:1034 42.
Pike JW, Gooz LL, Haussler MR. Biochemical evidence for 1,25dihydroxyvitamin D receptor macromolecules in parathyroid, pancreatic pituitary and placental tissues. Life Sci 1979;26:40714.
Sandgren ME, Bronnegard M, DeLuca HF. Tissue distribution of the
1,25-dihydroxyvitamin D3 receptor in the male rat. Biochem Biophys
Res Commun 1991;181:611 6.
Darwish HM, DeLuca HF. Identification of a transcription factor that
binds to the promoter region of the human parathyroid hormone gene.
Arch Biochem Biophys 1999;365:12330.
Slatopolsky E, Gonzalez E, Martin K. Pathogenesis and treatment of
renal osteodystrophy. Blood Purif 2003;21:318 26.
Yang S, Smith C, Prahl JM, DeLuca HF. Vitamin D deficiency suppresses cell-mediated immunity in vivo. Arch Biochem Biophys 1993;
303:98 106.
Yang S, Smith C, DeLuca HF. 1,25-Dihydroxyvitamin D3 and 19-nor1,25-dihydroxyvitamin D2 suppress immunoglobulin production and
thymic lymphocyte proliferation in vivo. Biochim Biophys Acta 1993;
1158:269 86.

49. Cantorna MT, Hayes CE, DeLuca HF. 1,25-Dihydroxyvitamin D3 reversibly blocks the progression of relapsing encephalomyelitis, a model
of multiple sclerosis. Proc Natl Acad Sci USA 1996;93:7861 4.
50. Zella JB, DeLuca HF. Vitamin D and autoimmune diabetes. J Cell
Biochem 2003;88:216 22.
51. Lemire JM, Ince A, Takashima M. 1,25-Dihydroxyvitamin D3 attenuates the expression of experimental murine lupus of MRL/1 mice. Autoimmunity 1992;12:143 8.
52. Cantorna MT, Munsick C, Bemiss C, Mahon BD. 1,25-Dihydroxycholecalciferol prevents and ameliorates symptoms of experimental murine inflammatory bowel disease. J Nutr 2000;130:2648 52.
53. Cantorna MT, Hayes CE, DeLuca HF. 1,25-Dihydroxyvitamin D3 prevents and ameliorates symptoms in two experimental model of human
arthritis. J Nutr 1998;128:68 72.
54. Griffin MD, Lutz W, Phan VA, Bachman LA, McKean DJ, Kumar R.
Dendritic cell modulation by 1,25-dihydroxyvitamin D3 and its analogs: a vitamin D receptor-dependent pathway that promotes a persistent
state of immaturity in vitro and in vivo. Proc Natl Acad Sci USA 2001;
98:6800 5.
55. Hullett DA, Cantorna MT, Redaelli C, et al. Prolongation of allograft survival by 1,25-dihydroxyvitamin D3. Transplantation 1998;66:824 8.
56. Sicinski RR, Prahl JM, Smith CM, DeLuca HF. New 1,25-dihydroxy19-norvitamin D3 compounds of high biological activity: synthesis and
biological evaluation of 2-hydroxymethyl, 2-methyl and 2-methylene
analogs. J Med Chem 1998;41:466274.
57. Yamamoto H, Shevde NK, Warrier A, Plum LA, DeLuca HF, Pike JW.
2-Methylene-19-nor-(20S)-1,25-dihydroxyvitamin D3 potently stimulates gene-specific DNA binding of the vitamin D receptor in osteoblasts. J Biol Chem 2003;278:31756 65.
58. Shevde NK, Plum LA, Clagett-Dame M, Yamamoto H, Pike JW, DeLuca HF. A novel potent analog of 1,25-dihydroxyvitamin D3 selectively
induces bone formation. Proc Natl Acad Sci USA 2002;99:1348791.
59. Plum LA, Prahl JM, Ma X, et al. Biologically active noncalcemic analogs of 1,25-dihydroxyvitamin D with an abbreviated side chain containing no hydroxyl. Proc Natl Acad Sci USA 2004;101:6900 4.
60. Binkley N, Krueger D, Cowgill CS, et al. Assay variation confounds the
diagnosis of hypovitaminosis D: a call for standardization. J Clin Endocrinol Metab 2004;88:31527.
61. Shepard RM, DeLuca HF. Plasma concentrations of vitamin D3 and its
metabolites in the rat as influenced by vitamin D3 or 25-hydroxyvitamin
D3 intakes. Arch Biochem Biophys 1980;202:4353.

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44.

DELUCA